This invention relates to a method of oxidizing long straight-chain alcohols and aromatic alcohols to their corresponding aldehyde using a methanol induced alcohol oxidase of methyltrophic yeasts in a biphasic reaction media. More specifically, this invention relates to the use of whole cells of Pichia pastoris in a two-phase reaction fluid to produce benzaldehyde, hexanal, heptanal, octanal, nonanal, decanal, undecanal, phenyl acetaldehyde or hydrocinnamaldehyde and, secondly to the improvements in the process which can be achieved through the use of a purified alcohol oxidase in a two phase reaction system.
Growth of some methyltrophic yeasts of the genera Pichia, Hansenula, Candida and Torulopsis on limiting amounts of methanol results in the synthesis of sub-cellular organelles known as peroxisomes. These organelles contain alcohol oxidase and catalase, two enzymes which act in concert to oxidize methanol to formaldehyde, the first step in the pathway which will ultimately result in complete degradation of methanol. When whole cells of the yeast are exposed to alcohols other than ethanol, the alcohol to aldehyde oxidation step is carried out, however, further degradation of the aldehyde is prevented by the substrate specificity of the second enzyme in the pathway, formaldehyde dehydrogenase. For this reason, the aldehyde product accumulates and the non-growing whole cells can be used as a biocatalyst in a process designed to produce a range of oxidized products. (W.D. Murray et al, Canadian Patent Application 588,365, 16 Jan. 1989.)
In the above cited Canadian patent application, whole cells of P. pastoris were used in an aqueous system to oxidize saturated and unsaturated straight chain and aromatic primary alcohols to their corresponding aldehydes. The rate of oxidation decreases with increasing chain length and with the addition of branches to the alcohol. Although a decrease in enzyme efficiency might be expected as one moves farther from the natural substrate of the enzyme, the problem is compounded by the decreasing water solubility of the reactants and products as the chain length increases. Particularly in reaction systems in which substrate and/or end product inhibition is severe, precipitation of either alcohol or aldehyde from solution can have extremely adverse effects on process kinetics because of the exposure to the enzyme to essentially pure inhibitor. The use of an essentially non-aqueous reaction medium in which both reactants and products are more soluble has been found to be a means, to improve the yield of the desired product.
The prior art demonstrates that methanol-induced alcohol oxidase from P. pastoris, or other methylotrophic yeasts, show a broad substrate specificity for short chain normal primary aliphatic alcohols. (R. Couderc and J. Baratti, Agric. Biol. Chem. 44: 2279-2289, 1980; R.N. Patel et al, Arch. Biochem and Biophys. 210: 481-488, 1981; Y. Tani et al, Agric. Biol. Chem. 36: 76-83, 1972; and U.S. Pat. No. 4,619,898.) Patel et al (op.cit.) determined that purified alcohol oxidase from P. pastoris was unable to oxidize secondary alcohols, diols, aromatic alcohols, cyclohexanol, tert butanol, 2-amino-ethanol or 2-amino-1-propanol. The concept of carrying out enzymatic reactions by means of the use of biphasic aqueous-organic systems is not novel. For example, Canadian Patent No. 1,005,771 discloses the use of laccase, steroid dehydrogenase lipase, lactic dehydrogenase and alcohol dehydrogenase in a biphasic system. However, based on the reports in the prior art which state that alcohol oxidase is unable to oxidize long chain alcohols, it was quite unexpected that use of a biphasic system would extend the known substrate range of alcohol oxidase to include benzyl alcohol, heptanol, octanol, nonanol, decanol, undecanol, phenethyl alcohol and 3-phenyl-1-propanol.
U.S. Pat. No. 3,880,739 discloses the oxidation of hydrophobic paraffins or alcohols to aldehydes of corresponding chain lengths by use of an aqueous suspension of a crude enzyme mixture in a hydrocarbon phase. In particular the enzymes were extracted from Pichia yeast cells which had been grown on an n-tetradecane substrate. It has recently been reported by G.D. Kemp et al (Appl. Microbiol. Biotechnol. 29: 370-374, 1988) that alkane grown Candida tropicalis produced an alcohol oxidase quite distinct from the alcohol oxidase previously reported for methylotrophic yeasts. The specificity of this alkane-induced enzyme for saturated primary alcohols differed from the methanol-induced enzyme. The optimum activity of the alkane-induced alcohol oxidase was towards dodecanol, with virtually no activity against the short chain (C.sub.2 -C.sub.6) alcohols. The method disclosed in U.S. Pat. No. 3,880,739 is directed to an alkane-induced enzymes for the oxidation of long-chain alcohols. One aspect of the present invention is directed to a strain of P. pastoris induced by methanol to produce a different enzyme which was, prior to this invention, not known to oxidize longchain substantially water-insoluble alcohols.